TaqMan Protein Assays

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1 QUICK REFERENCE CARD TaqMan Protein Assays Note: For safety and biohazard guidelines, refer to the Safety section in the TaqMan Protein Assays Sample Prep and Assays Protocol (Part no ). For every chemical, read the Safety Data Sheet (SDS) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Workflow Lysate dilutions Binding reaction Ligation reaction Protease reaction PCR Analysis Serially dilute reference cell Initial dilution of 1: or 1:10 Serially dilute test cell Initial dilution of 1: or 1:10 Prepare Assay Probe solution 1 µl Assay Probe A 1 µl Assay Probe B 16 µl Assay Probe Dilution Buffer Plate binding reactions µl/well Assay Probe solution µl/well Diluted Dilute ligase µl DNA Ligase µl Ligase Dilution Buffer Prepare ligation solution 600 µl Ligation Reaction Buffer + 10,908 µl Water + 1 µl Diluted DNA Ligase Stop ligation 4µL Protease µl PBS Add µl/well Incubate 37 C, 10 min 95 C, 5 min Prepare PCR mix 100 µl TaqMan Protein Assays Fast Master Mix 10 µl Universal PCR Assay Plate PCRs 11 µl/well PCR mix 9 µl/well proteasetreated ligation products Analyze with automatic baseline and a threshold cycle of 0. Export the C T data Incubate 37 C, 60 min Plate ligation reactions 96 µl/well ligation solution Perform real-time PCR Incubate 37 C, 10 min Prepare dilutions of each cell 1 Determine the starting concentrations and minimum dilutions Sample preparation kit Sample Recommended concentration Cell count Protein concentration Minimum initial dilution Recommended highest amount per well ( µl of sample/well) Protein Expression Sample Preparation Kit Protein Quant Sample Lysis Kit Other sample prep kits (Refer to the Protocol for a list of kits.) NTERA and Raji Protein Expression Lysate Control Kits Cell s 500 cells/µl NA 1: 500 cells 500 cells/µl 50 ng/µl 1: cells or 50 ng 500 cells/µl 50 ng/µl 1: cells or 50 ng Tissue s NA 5000 ng/µl 1: ng 500 cells/µl (provided) NA 1: 500 cells Recombinant protein in buffer NA 100,000 pg/ml None 00 pg

2 Prepare the cell dilutions IMPORTANT! Prepare the cell dilutions on ice. The diluted s must be used on the day of preparation. Do not store the diluted s overnight. A B C D Reference cell a. Thaw the appropriate buffer and s on ice, then place a 96-well reaction plate on ice. b. Prepare -fold serial dilutions of the reference and test cell s: E F G H Test cell Initial dilution Step Action Reaction plate well number Reference cell Test cell 1: 1 Add 1 µl of Cell Resuspension Buffer (or Lysate Dilution Buffer). Add 1 µl of cell. Pipet up and down several times to mix the sample. 3 Continue to transfer 1 µl of cell from the previous dilution well to the next dilution well, pipetting up and down several times. The buffer formulations in each kit are identical; the buffers can be used interchangeably. c. Briefly centrifuge the plate, then place the plate back on ice. A1 through A1 A1 A11 is the last well; A1 contains no 1:10 1 Add 18 µl of Lysate Dilution Buffer. A1 E1 Add 1 µl of Lysate Dilution Buffer. A through A1 3 Add µl of cell. Pipet up and down several times to mix the sample. 4 Continue to transfer 1 µl of cell from the previous dilution well to the next dilution well, pipetting up and down several times. A1 A11 is the last well; A1 contains no E1 through E1 E1 E11 is the last well; E1 contains no E through E1 E1 E11 is the last well; E1 contains no

3 Perform the binding reaction 1 Prepare the Assay Probe solution IMPORTANT! Prepare the Assay Probe solution no more than 0 minutes before performing the binding reaction. a. Place the following reagents on ice: Assay Probe Dilution Buffer, Assay Probe A, Assay Probe B. b. When thawed, gently mix the Assay Probe Dilution Buffer. Gently mix the Assay Probe A and Assay Probe B solutions. c. Combine the components listed below in the order indicated. Mix gently, then centrifuge the tube. Place on ice. Order to combine 1 Assay Probe Dilution Buffer, 1 16 µl Assay Probe A, 0 1 µl 3 Assay Probe B, 0 1 µl Total volume of Assay Probe solution 40 µl The total volume is sufficient for preparing one 96-well reaction plate. Prepare and incubate the binding reaction plate a. Place a 96-well reaction plate on ice. IMPORTANT! Keep the reaction plate on ice while you add the reaction components. b. Add µl of the Assay Probe solution to each well of the reaction plate. c. Transfer µl of the diluted reference cell from wells A1 through A1 of the dilution plate to the corresponding wells in row A of the binding reaction plate. Repeat for rows B through D of the binding reaction plate. d. Transfer µl of the diluted test cell from wells E1 through E1 of the dilution plate to the corresponding wells in row E of the binding reaction plate. Repeat for rows F through H of the binding reaction plate. e. Seal the binding reaction plate with a MicroAmp Clear Adhesive Film, then briefly centrifuge the plate. f. Incubate the sealed reaction plate using the thermal-cycling conditions below. IMPORTANT! To prevent condensation and evaporation, you must use a compression pad and a heated cover when using the thermal cycler. Failure to do so will result in highly variable data. Stage Temperature ( C) Time HOLD 37 C 60 minutes HOLD 4 C Keep the binding reactions at 4 C until you are ready to open the binding reaction plate. Applied Biosystems recommends that you proceed to Perform the ligation and protease reactions on page 4 within 15 minutes. 3

4 Perform the ligation and protease reactions IMPORTANT! Keep all reagents on ice when not in use. Do not allow the tubes to warm to room temperature. Keep the reaction plates on ice during reagent transfers. 1 Perform the ligation reaction a. Thaw or place the following components on ice: 500 DNA Ligase, 1 Ligase Dilution Buffer, 0 Ligation Reaction Buffer, 1 PBS (ph 7.4), 100 Protease. b. Dilute the DNA Ligase: Combine the components listed below. Mix gently, then place on ice. IMPORTANT! Prepare fresh diluted ligase for each experiment. DNA Ligase, 500 µl Ligase Dilution Buffer, µl Total volume of diluted DNA Ligase 1000 µl c. Prepare the ligation solution: Combine the components listed below. Invert the tube to mix, then place on ice. 1 reaction 1 plate Ligation Reaction Buffer, µl 600 µl Nuclease-free water 90.9 µl 10,908 µl Diluted DNA Ligase 0.1 µl 1 µl Total volume of ligation solution 96.0 µl 11,50 µl d. Remove the binding reaction plate from the thermal cycler, remove the MicroAmp Clear Adhesive Film, then place the plate on ice. e. Add 96 µl of the ligation solution to each well in the binding reaction plate. Pipet up and down once to mix. f. Reseal the ligation reaction plate with a new adhesive film, then briefly centrifuge the plate. g. Incubate the sealed reaction plate using the thermal-cycling conditions below. IMPORTANT! To prevent condensation and evaporation, you must use a compression pad and a heated cover when using the thermal cycler. Failure to do so will result in highly variable data. Step Cycle number Temperature ( C) Time Reaction volume Ligation 1 37 C 10 minutes Default Cooling 1 4 C Up to 10 minutes Default You can omit the protease step if you proceed immediately to Perform the real-time PCR on page 6. Otherwise, continue with Perform the protease reaction on page 5. 4

5 Perform the protease reaction a. Dilute the protease: 1. Briefly vortex the protease to mix the solution.. Combine the components listed below. Mix gently, then briefly centrifuge to spin the liquid to the tube bottom. Place on ice. Protease, µl 1 PBS, ph µl Total volume of diluted protease solution 400 µl b. Remove the ligation reaction plate from the thermal cycler, remove the MicroAmp Clear Adhesive Film, then place the plate on ice. c. Add µl of the diluted protease to each well of the ligation reaction plate. Note: No mixing is required. The protease will diffuse throughout the samples during the 10- minute incubation. d. Reseal the reaction plate with a new adhesive film. e. Incubate the sealed reaction plate using the thermal-cycling conditions below. IMPORTANT! To prevent condensation and evaporation, you must use a compression pad and a heated cover when using the thermal cycler. Failure to do so will result in highly variable data. Step Terminate ligation Inactivate protease Cycle number Temperature ( C) Time 1 37 C 10 minutes Default 1 95 C 5 minutes Default HOLD 1 4 C Hold Default f. Remove the reaction plate from the thermal cycler and place it on ice. Reaction volume Proceed to Perform the real-time PCR on page 6 OR store the protease-treated ligation products at 4 C for up to 3 days, or at 0 C for up to weeks. 5

6 Perform the real-time PCR IMPORTANT! Keep all reagents on ice when not in use. Do not allow the tubes to warm to room temperature. Keep the reaction plates on ice during reagent transfers. 1 Prepare the PCR mix a. Thaw the Universal PCR Assay on ice. b. Combine the components listed below. Mix gently, then briefly centrifuge to spin the liquid to the tube bottom. Place on ice. 1 reaction 1 plate Fast Master Mix, 10 µl 100 µl Universal PCR Assay, 0X 1 µl 10 µl Total volume of PCR mix 11 µl 130 µl A B C D E F G H Reference cell Prepare the PCR plate a. Place a PCR plate on ice, then add 11 µl of the PCR mix to each well. Test cell A B C D E F G H b. Remove the MicroAmp Clear Adhesive Film from the protease reaction plate, then place the plate on ice. c. Transfer 9 μl of the protease-treated ligation product from each well of the protease reaction plate to each corresponding well of the PCR plate. When transferring, pipet up and down once to mix. d. Seal the PCR plate with a MicroAmp Optical Adhesive Film, then briefly centrifuge the plate. For the 7900HT/7900HT Fast system with a 96-Well Block Module and automation accessory, place a MicroAmp Snap-On Optical Film Compression Pad on top of the plate. IMPORTANT! Proceed immediately to Run the PCR plate on page 7. 6

7 3 Run the PCR plate In your real-time PCR system software, create a plate document/experiment for the run, using the setup information below. Load the PCR plate into your real-time PCR instrument, then start the run. System StepOnePlus 7500 Fast 7900HT Fast 7900HT Software Analyze the data StepOne Software v1.0 or later SDS Software v1.4 or later 7500 Software v.0 or later SDS Software v.1 or later Template cdna cdna Run type Fast Standard Reaction plate Fast 96-well Fast 96-well, Standard 96-well, or 384-well SDS Software v.0 or later Standard 96-well or 384- well Sample volume 0 µl 0 µl Detectors/ targets Reporter: FAM dye Quencher: Non-fluorescent Reporter: FAM dye Quencher: Non-fluorescent Ramp speed/ mode Fast Fast or Standard Standard Experiment type Select an experiment type that will generate C T values, such as Absolute Quantitation or Standard Curve. Tasks and You do not need to assign tasks or quantities. quantities Analysis settings Threshold cycle (C T ): 0.; Baseline: Automatic Method Using the real-time PCR system software Using a spreadsheet program Using the ProteinAssist Software Procedure 1. View the amplification plots for the entire reaction plate.. Analyze the plate run using a threshold cycle (C T ) setting of 0. and automatic baseline. 1. Export the results from the instrument software to a spreadsheet program.. Calculate the average C T values for each sample. 3. Calculate the ΔC T values for each sample: AvgC T (NPC) AvgC T (sample) 4. Plot the ΔC T values vs. cell input on a semi-log graph. To determine the relative protein expression of a given target between different samples: 1. Download the ProteinAssist Software from: Refer to the Getting Started Guide for instructions on how to import and analyze C T data. (To open the Getting Started Guide, click the Help tab in the software.) 7

8 For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Limited Use Label License: Research Use Only The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California Limited Use Label License: Product Disclaimer The products referred to in this Quick Reference Card may be covered by one or more Limited Use Label License(s). Please refer to the respective product documentation or the Applied Biosystems website under for the comprehensive license information. By use of these products, the purchaser accepts the terms and conditions of all applicable Limited Use Label Licenses. These products are sold for research use only, and are not intended for human or animal diagnostic or therapeutic uses unless otherwise specifically indicated in the applicable product documentation or the respective Limited Use Label License(s). For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California Trademarks The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TaqMan is a registered trademark of Roche Molecular Systems, Inc. 011 Life Technologies Corporation. All rights reserved. Part Number Rev. B 0/011 Headquarters 5791 Van Allen Way Carlsbad, CA 9008 USA Phone Technical Resources and Support For the latest technical resources and support information for all locations, please refer to our Web site at